Numerous natural and synthetic proteins are used in diagnostic and therapeutic applications and many others are in development or in clinical trials. Current methods of protein production include isolation from natural sources and recombinant production in cell culture. Because of the complexity, high cost and known problems of these methods such as batch failure, efforts have been underway to develop alternatives. For example, methods for producing exogenous proteins in the milk of pigs, sheep, goats, and cows have been reported. These approaches have certain limitations, including long generation times between founder and production herds, extensive husbandry and veterinary costs. Proteins are also being produced using milling and malting processes from barley and rye. However, plant post-translational modifications substantially differ from vertebrate post-translational modifications, which can have a negative effect on the function of the exogenous proteins such as pharmaceutical proteins.
The avian oviduct can also serve as a bioreactor. Successful methods of modifying avian genetic material such that high levels of exogenous proteins are secreted in the oviduct and packaged into eggs allows inexpensive production of large amounts of protein. Several advantages of this approach are: a) short generation times (24 weeks) and rapid establishment of transgenic flocks via artificial insemination; b) readily scaled production by increasing flock sizes to meet production needs; c) post-translational modification of expressed proteins; d) automated feeding and egg collection; e) naturally aseptic egg-whites; and f) reduced processing costs due to the high concentration of protein in the egg white.
Recent developments in avian transgenesis have allowed the modification of avian genomes for exogenous protein production. Germ-line transgenic chickens may be produced by injecting replication-defective retrovirus into the subgerminal cavity of chick blastoderms in freshly laid eggs. See, for example, U.S. Pat. No. 7,511,120, issued Mar. 31, 2009, the disclosure of which is incorporated in its entirety herein by reference; issued U.S. Pat. No. 7,338,654, issued Mar. 4, 2008, the disclosure of which is incorporated in its entirety herein by reference; and US patent publication No. 2008/0064862 published Mar. 13, 2008, the disclosure of which is incorporated in its entirety herein by reference.
Limitations of currently accepted methods of producing therapeutic proteins include the expense of producing the production facilities and batch failure. What is needed are improved methods of producing therapeutic or pharmaceutical proteins such as fusion proteins including novel and improved forms of CTLA4-Fc.